The goal of this research project is to investigate the intermolecular interactions and mechanisms of electron transfer between cytochrome c and cytochrome oxidase and reductase. Seim-synthetic analogs of cytochrome c will be constructed by combining cyanogen bromide fragments 1-65 and 81-104, prepared from natural cytochrome c, with synthetic peptides corresponding to residues 66-80, prepared by solid phase peptide synthesis. The latter sequence contains most of the amino acids which are thought to be mechanistically important, and thus, we plan to test various proposed mechanisms by replacing supposedly important amino acids with different amino acids which could not function in the same manner. Other synthetic analogs will be designed to investigate the relationship of the oxidation-reduction potential of cytochrome c to the conformational changes associated with changes of its oxidation state. Efforts will be made to determine which amino acids participate in the interactions of cytochrome c with cytochrome oxidase and cytochrome c reductase by introducing amino acid substitutions which weaken or inhibit these interactions. The chemical purity of the synthetic cytochrome c analogs will be carefully analyzed, and their oxidation-reduction potentials and their visible, ultraviolet and circular dichroism spectra will be compared with the corresponding properties of natural cytochrome c. The biological activities of the synthetic analogs will be determined in several different electron transport assay systems, including the Keilin-Hartree heart muscle preparation, and the related NADH-cytochrome c reductase and cytochrome oxidase systems. Our studies of the specific interactions of cytochrome c analogs with the oxidase and reductase complexes isolated from heart mitochondria should provide a more detailed understanding of the intricate relations among these important components of the respiratory electron transport chain, which produces the ATP required to sustain normal heart activity. BIBLIOGRAPHIC REFERENCES: Warme, P.K. and Scheraga, H.A., "Conformational Energy Reginement of Horse-Heart Ferricytochrome c", Biochemistry 14, 3509 (1975). Warme. P.K., "The Influence of Amino Acid Substitutions on the Conformational Energy of Cytochrome c", Biochemistry 14, 3518 (1975).